The present invention relates to compositions and methods for treating sperm for the purpose of modifying sperm function and the gender ratio in offspring of mammalian species. The invention is further directed to a method of using the composition to modify the functionality of mammalian sperm in general and more specifically, to increase sperm fertility for the purpose of enhancing conception. In addition, the invention can be used to modify the fertility of X-chromosome and Y-chromosome bearing sperm and using said sperm in the reproduction processes of artificial insemination (AI), in vitro fertilization (IVF), and embryo transfer (ET) for the purpose of modifying gender in mammals.
It is well documented in mammalian species that the X-chromosome contains a unique set of genes which are highly conserved across mammals as well as other vertebrates. One of the X-linked genes codes for the ubiquitous enzyme, glucose-6-phosphate dehydrogenase (G6PDH). G6PDH is a pivotal enzyme in glucose metabolism and is the primary regulator of the hexose-mono phosphate shunt (HMS), also known as the pentose phosphate shunt (PPP). The main function of the HMS is to produce NADPH, which is necessary for reduction-oxidation reactions and to form ribose-5-phosphate for nucleic acid synthesis.
G6PDH also play an important role in glucose oxidation via glycolysis, a primary source of cellular energy. Glucose metabolism is implicated in the fertilization process in many mammalian species. It is well accepted that glucose metabolism through glycolysis provides energy to sperm. However, the role of glucose metabolism through the hexose-monophosphate shunt (HMS) in spermatozoa is not understood. The existence of an HMS pathway in mouse and human sperm has been documented, but not in other species including ram or bull sperm. However, techniques used for these early studies have come into question and the evidence does not disprove the existence of the HMS in sheep or cattle sperm.
The implications of a functioning HMS in sperm, as found in human and mouse sperm, suggests that sperm need to produce NADPH via the HMS to achieve fertilization. Since NADPH metabolism has been implicated in sperm motility and fertilization, it also suggests that the HMS is a key metabolic pathway in sperm capacitation, the acrosome reaction, and oocyte fusion.
While investigating the role of G6PDH and the HMS as a method to sex mouse embryos, I first used the phenoxazine compound, brilliant cresol blue (BCB) in living embryos to detect and semi-quantify G6PDH activity and successfully transplant these embryo to produce normal living offspring. This study demonstrated that BCB and its metabolites were relatively non-toxic in the early-staged mouse embryos. My studies with bovine embryos revealed similar results.
Prior art teaches that the HMS in bovine oocytes, the phenoxazine/phenothiazine class of compounds can be used to increase glucose oxidation specifically through the Hexose Monophosphate Shunt (HMS) and that BCB stimulates a 15-fold increase in oxidation of glucose through the HMS as well as an increased level of glucose metabolism through the HMS in female embryos.
I also first used the electron transfer agent, NADPH oxioreductase (Diaphorase) to further amplify the HMS in living mouse and cattle embryos No patents concerning the use of phenoxazine/phenothiazine and/or electron transfer agents in living gametes or embryos have arisen from these studies.
It is well documented that early staged female embryos exhibit preferential metabolism of glucose via the HMS. This is due to the presence of two X-chromosomes and elevated levels of G6PDH. However, there is no prior art concerning differential glucose metabolism of X-chromosome and Y-chromosome bearing sperm. Concerning this invention, it is postulated that mammalian sperm (like embryos) retain a HMS pathway and metabolize glucose and more specifically, G-6-P, through this pathway to produce NADPH. It is also postulated that the X-chromosome bearing (female) sperm can preferentially oxidize G-6-P through the HMS pathway due to higher levels of X-linked enzyme, G6PDH, relative to the Y-chromosome bearing (male) sperm. The reason for the sex differential in oxidation of G-6-P is speculative but could have arisen as a mechanism to regulate sex allocation in natural populations. Given this assumption, the X-chromosome bearing (female) sperm is thought to readily oxidize glucose-6-phosphate through the HMS pathway. When sperm are exposed to a chromo-phenoxazine compound, the HMS pathway is promoted and amplified to produce large amounts of NADPH. This amplification occurs until the phenoxazine compound is completely reduced to the leuco-phenoxazine form or the reaction is inhibited due to substrate depletion. With the addition of excess pentose substrate (preferentially G-6-P), the reaction continues until complete phenoxazine reduction. The other major component and potentially rate-limiting to the reaction is NADP+. This cofactor is needed in the first and third steps of the HMS pathway. Endogenous electron transfer agents such as NADPH dehydrogenase or NADPH oxidase are unlikely to be available in sustainable titers. However, with the addition of an exogenous electron transfer agent such as NADPH oxioreductase (usually coupled with a cofactor flavin mononucleotide), the NADPH is oxidized to NADP+ during the transfer of electrons, i.e., the reduction of the chromo-phenoxazine. The NADP+ is then available for reuse in the HMS pathway. Under these conditions, the oxidation of the available pentose sugar, G-6-P, can continue until the substrate becomes limiting or the phenoxazine become completely reduced. The result of the amplification of the HMS are large amounts of reducing power in the form of NADPH which are correlated with increased sperm function, motility, and fertilization capacity. Other substrate precursors such as ribose-5-phosphate, lactate, pyruvate, and NADH would also be generated in high titers and would be available for increased rates of glycolysis, ATP production, and respiration. These precursors could result in increased sperm function, higher motility, and more specifically, increased fertilization capacity for of the X-chromosome bearing (female) sperm.
Another observation I have made is that when sperm are exposed to Brilliant Cresol Blue, there is reduced sperm motility and fertility. This loss of motility is postulated to be due to a quenching of the HMS, a shut-down of oxidative phosphorylation, a loss of respiration potential and reduced ATP production. The result is a potential loss of fertilization capacity and sperm viability. In the composition of this invention, it is postulated that the Y-chromosome bearing (male) sperm are exposed to phenoxazine compounds without the benefit of G6PDH. As a result, the male sperm cannot oxidize the available G6P through the HMS pathway and are unable to generate large quantities of NADPH necessary to reduce the chromo-phenoxazine. This in turn results in reduced motility and fertility of the Y-chromosome bearing (male) sperm.
In addition, NADPH oxioreductase may have the opposite effect on the Y-chromosome bearing sperm than what occurs in the X-chromosome bearing sperm. One possibility is that NADPH oxioreductase inhibits glycolysis. This inhibition may be competitive, related to redox potential, or may be due to the high level of specificity of the NADPH oxioreductase enzyme for the NADH that is generated in glycolysis. While the exact mechanism is unknown, it has been observed that the Y-chromosome bearing (male) sperm, when exposed to the composition of this invention, have reduced fertility.
Patent documents of interest concerning methods to preferentially modify sperm function include: U.S. Pat. Nos. 4,191,749, 4,191,749, 4,999,283, 4,788,984, 6,627,655, and 20070166694.
Patent documents of interest concerning the use of phenoxazine BCB in living tissue are limited to in vitro assays, dye indicators, and staining methodology. These include: U.S. Pat. Nos. 6,790,411, 6,867,015, 6,967,015, 6,420,128, and 4,622,395.
Patent documents of interest related to methods of modifying sex ratio in mammals by cell sorting technology include: U.S. Pat. Nos. 6,524,860, 6,372,422, 6,149,867, 6,071,689, and 5,135,759.
Patent documents of interest related to modifying sex ratio in mammals by antigen or antibody sorting include: U.S. Pat. Nos. 6,489,092, 6,153,373, 5,660,997, and 5,439,362.